A proper understanding of the mechanism of oxygen supply to tissue depends on a detailed comprehension of the function of hemoglobin. The molecular basis of the cooperative oxygen binding and the allosteric behavior of human hemoglobin will be investigated by a new heme-spin- label technique. The efficiency of normal, abnormal, and modified hemoglobins in supplying tissue oxygen will be evaluated by a special procedure employing perfused organs, particularly the heart. Spin-labels attached to different positions of the porphyrin ring in hemoproteins provide important structural information on the local environment of the label in the dissolved protein molecule under physiological conditions and during functional changes. The mechanisms of cooperative oxygen binding to hemoglobin will be studied with spinlabeled mixed-state hemoglobins to which one type of subunit contains spin-labeled ferric hemes while the other has native ferrous hemes. The effect of oxygen binding to one type of subunit on the heme environment of the other type can be studied by simulataneous measurements of electron paramagnetic resonance and optical spectra in an apparatus recently constructed by the applicant for the automatic determination of oxygen dissociation curves. The effect of pH change, as well as 2,3-diphosphoglycerate on native, abnormal, and spin-labeled hemoglobins will also be examined. The heme-spinlabel method will be further extended to the elucidation of the structure-function relationships of abnormal hemoglobins and hemoglobins from other species. As shown in the attached progress report, a number of very interesting results have been obtained by the heme-spin-label method. Since the hemglobin has the same optical and oxygen binding properties as those of normal hemoglobin, this hemoglobin was used as an excellent tool for studying the mechanism of oxygen binding to hemoglobin. Another type of heme-spin-label hemoglobin, in which a spinlabel is attached at one of the vinyl groups, will be used for studying the mode of important interaction between heme and amino acid residues of hemoglobin.